Purification and properties of an alpha-D-xylosidase from Aspergillus niger

Two components of alpha-D-xylosidase (alpha-D-xylosidase I and II) were detected in the culture filtrate of Aspergillus nigher grown in a medium containing Sanzyme 1000-treated Glyloid 2A. The major component (alpha-D-xylosidase I) was purified to an electrophoretically pure state. The purified enzyme showed approximately 540-fold increase in specific activity over the original culture filtrate. The purified enzyme was shown to be an oligomeric protein consisting of four subunits, each of which had a molecular weight of 123,000. The enzyme showed the highest activity at pH 2.5-3.0 and 45 degrees C, and was stable in the pH range from 3.0 to 7.0 and at the temperatures up to 60 degrees C. The isoelectric point of this enzyme was pH 5.6. The purified enzyme was highly specific for p-nitrophenyl alpha-D-xylopyranoside and isoprimeverose (6-O-alpha-D-xylopyranosyl-D-glucopyranose). The apparent Km and Vmax values of the enzyme for p-nitrophenyl alpha-D-xylopyranoside and isoprimeverose were 10.5 mM and 40.8 mumol/min/mg protein, and 2.2 mM and 30 mumol/min/mg protein, respectively. The purified enzyme could also split off the alpha-D-xylopyranosyl residue on the non-reducing terminal of the backbone of oligoxyloglucans such as alpha-D-xylopyranosyl-(1----6)-beta-D-glucopyranosyl- (1----4)-[(alpha-D-xylopyranosyl-(1----6)-]-beta-D-glucopyranosyl- (1----4)-] 2-D-glucopyranose.     

Purification and characterization of beta-N-acetylhexosaminidase from the ascidian, Halocynthia roretzi

beta-N-Acetylhexosaminidase [EC 3.2.1.30] was purified 820-fold from the viscera of Halocynthia roretzi by Sephadex G-200 gel filtration and chromatography on columns of DEAE-Sephadex and CM-Sephadex. The final preparation was sufficiently free from alpha-N-acetylglucosaminidase, alpha-N-acetylgalactosaminidase, alpha- and beta-glucosidases, alpha- and beta-galactosidases, alpha- and beta-mannosidases, and alpha-L-fucosidase, and gave one protein band on disc gel electrophoresis. Two different molecular weight forms which depended upon the pH were observed on Sephadex gel filtration. At pH 7.0, a species with a molecular weight of 170,000 was observed, whereas at pH 4.5, an enzyme of 330,000 daltons was seen. The enzyme was active at pH 4.5 but inactive at pH 7.0. The optimum pH and the Km were pH 4.2 and 1.9 mM for p-nitrophenyl beta-N-acetylglucosaminide and pH 4.0 and 0.9 mM for p-nitrophenyl beta-N-acetylgalactosaminide. The terminal beta-N-acetylhexosamine of glycolipids such as globoside I, GM2, and asialo GM2 was cleaved by the ascidian beta-N-acetylhexosaminidase though GM2 was less susceptible to the enzyme.     

Cationic form of beta-galactosidase in the germinating seeds of Vigna sinensis (Linn) Savi

The cationic form of beta-galactosidase (EC 3.2.1.23) from the germinating seeds of Vigna sinensis has been separated from its other isoforms by DEAE-cellulose (DE-52) column chromatography and further purified by gel filtration and affinity chromatography. Polyacrylamide gel electrophoresis of the purified enzyme imparted a single protein band. The molecular mass of the enzyme as determined by Sephadex G-150 gel filtration is 58,800 Da. The optimum temperature and the optimum pH are 60 degrees C and 4.5, respectively. Most of the metal ions tested were inhibitory to the enzyme activity. The enzyme has Km for p-nitrophenyl beta-D-galactoside and o-nitrophenyl beta-D-galactoside of 0.56 and 2.0 mM, respectively. The Ki values of galactose and lactose are 2.4 and 70.0 mM, respectively. The energy of activation of PNPG for the enzyme is 10.3 kcal/mol.     

Purification and properties of mitochondrial malate dehydrogenase from unfertilized eggs of the sea urchin, Anthocidaris crassispina

Purification and characterization of mitochondrial malate dehydrogenase [EC 1.1.1.37] from unfertilized eggs of the sea urchin, Anthocidaris crassispina, are described. The purification method consisted of dextran sulfate fractionation, Blue Dextran Sepharose chromatography, Phenyl-Sepharose hydrophobic chromatography and DEAE-cellulose chromatography. The enzyme was purified 771-fold with a 7% yield from the crude extract. The purified enzyme appeared homogeneous on polyacrylamide gel electrophoresis under both native and denatured conditions. After incubation at 45 degrees C for 50 min, the enzyme lost about 90% of its activity. In the presence of NADH, however, the enzyme was protected against the heat denaturation. The native enzyme had a molecular weight of about 65,000 and probably consisted of two identical subunits. In the reduction of oxaloacetate with NADH, a broad optimum pH ranging from 8.2 to 9.4 was found with 50 mM Tris-HCl and glycine-NaOH buffers. Sodium phosphate buffer apparently activated the enzyme. The apparent Km values for oxaloacetate and NADH were 19 microM and 30 microM, respectively. The optimum pH for malate oxidation with NAD+ was 10.2 in 50 mM NaHCO3-Na2CO3 buffer. The apparent Km values for malate and NAD+ were 7.0 mM and 0.6 mM, respectively. Zinc ion, sulfite ion, p-chloromercuriphenylsulfonate and adenine nucleotides strongly inhibited the enzyme.     

Human lysosomal beta-glucosidase: purification by affinity chromatography

Two Sepharose-bound substrate analogs, 6'-aminohexanoyl-(2-N-sphingosyl-O-beta-D-glucoside) and 6'-aminohexyl-dodecanedioyl-1-(2-N-sphingosyl-1-O-beta-D-glu coside), were synthesized and used sequentially for the affinity purification of lysosomal beta-glucosidase (N-acyl-sphingosyl-1-O-beta-D-glucoside:glucohydrolase, EC 3.2.1.45). The capacities of these nondegradable affinity supports were 0.1 and 0.15 mg enzyme/ml settled gel, respectively. The purified enzyme had a specific activity of 75 mumol min-1 mg-1. The preparation had a single protein band with a molecular weight of 67,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, evidencing its apparent homogeneity. Isoelectric focusing on granular gels revealed four molecular forms of the enzyme with pI values of 4.0, 4.5, 4.7, and 5.8 to 6.2. The purified enzyme hydrolyzed glucosyl ceramide and 4-methylumbelliferyl-beta-D-glucoside with Km and Vmax values of 0.6 and 2.5 mM, and 101 and 26.1 mumol min-1 mg-1, respectively. The enzyme also hydrolyzed octyl beta-glucoside, a linear mixed-type inhibitor of the enzyme. Binding constants (Ki) were determined for the inhibitors, sphingosyl-1-O-beta-D-glucoside (Ki = 20 microM) and its N-hexyl derivative (Ki = 0.3 microM). The enzyme had a half-life of 65 and 30 min at 50 degrees C and pH 5.0 or 6.0, respectively. In addition, two other classes of ligands were used for the purification of lysosomal beta-glucosidase, and their capacities and specificities were compared to those of the substrate analog affinity supports. These included (i) the alkyl amine inhibitors octylamine, decylamine, and tetradecylamine; and (ii) the inhibitors, 6-aminohexanoyl-beta-glucosylamine and aminododecanoyl-1-(2-N-sphingosyl-1-O-beta-D-glucoside). Compared to these other ligand columns, the substrate analog affinity supports had about 100- to 1000-fold greater capacities or afforded 8- to 40-fold greater purification of human lysosomal beta-glucosidase.     

Effect of ethanol on the redox state of the coenzyme bound to alcohol dehydrogenase studied in isolated hepatocytes.

Human lactase was isolated from solubilized small-intestinal brush-border membranes by a combination of chromatography on concanavalin A-Sepharose, Bio-Gel 1.5m and chromatofocusing, with a yield of approx. 1% and a 750-fold purification. The enzyme appeared to be homogeneous on SDS/polyacrylamide-gel electrophoresis under both reduced and non-reduced conditions, with an apparent Mr of approx. 170,000. On gel filtration, however, it displayed an apparent Mr of approx. 380,000. The protein had a pI of 4.8, as judged by the chromatofocusing experiment, and had a lactase activity whose optimum is at pH 6.0. In addition to the beta-galactosidase activity, the protein also hydrolysed to various extents cellobiose, phlorizin, p-nitrophenyl beta-D-galactoside, p-nitrophenyl beta-D-glucoside, o-nitrophenyl beta-D-galactoside and o-nitrophenyl beta-D-fucoside. Antisera had been raised against the purified enzyme in two rabbits. One of the antibody populations could inhibit the enzyme in a concentration-dependent manner. This antibody population was used to set up an antibody-bound Sepharose column for the use in an immunoaffinity purification of lactase from crude intestinal homogenate. A partially purified preparation of lactase could thus be obtained. The antibody population was also used to set up a radioimmunoassay for quantifying the enzyme. The competition assay could detect about 0.5 micrograms of lactase protein/ml.     

Purification and some properties of a novel alpha-L-fucosidase capable of acting on alpha-(1----6)-L-fucosidic linkages from Bacillus circulans M28

Two types of alpha-L-fucosidase (F-I and F-II), that differ in substrate specificity, were produced in the culture fluid by Bacillus circulans isolated from soil when the bacterium was cultivated on medium containing porcine gastric mucin. F-I was able to cleave the alpha-(1----2), alpha-(1----3), and alpha-(1----4)-L-fucosidic linkages in various oligosaccharides and glycoproteins, but not p-nitrophenyl alpha-L-fucoside, as previously reported [Y. Tsuji et al. (1990) J. Biochem. 107, 324-330]. F-II was purified from the culture fluid obtained with glucose medium by ammonium sulfate fractionation and various subsequent column chromatographies. The purified enzyme was found to be homogeneous on PAGE and its molecular weight was estimated to be approximately 250,000. The maximal activity was observed between pH 6.0 to 7.0, the stable pH range being 6.0 to 8.5. The enzyme specifically cleaved alpha-L-fucosidic bonds in low molecular weight substrates. The enzyme cleaved not only p-nitrophenyl alpha-L-fucoside, but also 2-fucosyllactose and 3-fucosyllactose. The enzyme was also able to act on the alpha-(1----6)-L-fucosidic linkages to N-acetylglucosamine in 6-O-alpha-L-fucopyranosyl-N-acetylglucosamine, and bi- and tetra-antennary oligosaccharides derived from porcine pancreatic lipase, which were not hydrolyzed by F-I.     

Phosphotyrosyl-specific protein phosphatase activity of a bovine skeletal acid phosphatase isoenzyme. Comparison with the phosphotyrosyl protein phosphatase activity of skeletal alkaline phosphatase

A partially purified bovine cortical bone acid phosphatase, which shared similar characteristics with a class of acid phosphatase known as tartrate-resistant acid phosphatase, was found to dephosphorylate phosphotyrosine and phosphotyrosyl proteins, with little activity toward other phosphoamino acids or phosphoseryl histones. The pH optimum was about 5.5 with p-nitrophenyl phosphate as substrate but was about 6.0 with phosphotyrosine and about 7.0 with phosphotyrosyl histones. The apparent Km values for phosphotyrosyl histones (at pH 7.0) and phosphotyrosine (at pH 5.5) were about 300 nM phosphate group and 0.6 mM, respectively, The p-nitrophenyl phosphatase, phosphotyrosine phosphatase, and phosphotyrosyl protein phosphatase activities appear to be a single protein since these activities could not be separated by Sephacryl S-200, CM-Sepharose, or cellulose phosphate chromatographies, he ratio of these activities remained relatively constant throughout the purification procedure, each of these activities exhibited similar thermal stabilities and similar sensitivities to various effectors, and phosphotyrosine and p-nitrophenyl phosphate appeared to be alternative substrates for the acid phosphatase. Skeletal alkaline phosphatase was also capable of dephosphorylating phosphotyrosyl histones at pH 7.0, but the activity of that enzyme was about 20 times greater at pH 9.0 than at pH 7.0. Furthermore, the affinity of skeletal alkaline phosphatase for phosphotyrosyl proteins was low (estimated to be 0.2-0.4 mM), and its protein phosphatase activity was not specific for phosphotyrosyl proteins, since it also dephosphorylated phosphoseryl histones. In summary, these data suggested that skeletal acid phosphatase, rather than skeletal alkaline phosphatase, may act as phosphotyrosyl protein phosphatase under physiologically relevant conditions.     

Pre-steady-state and steady-state kinetic analysis of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.

The complete time course of the hydrolysis of p-nitrophenyl phosphate catalyzed by the low molecular weight (acid) phosphotyrosyl protein phosphatase from bovine heart was elucidated and analyzed in detail. Burst titration kinetics were demonstrated for the first time with this class of enzyme. At pH 7.0, 4.5 degrees C, a transient pre-steady-state "burst" of p-nitrophenol was formed with a rate constant of 48 s-1. The burst was effectively stoichiometric and corresponded to a single enzyme active site/molecule. The burst was followed by a slow steady-state turnover of the phosphoenzyme intermediate with a rate constant of 1.2 s-1. Product inhibition studies indicated an ordered uni-bi kinetic scheme for the hydrolysis. Partition experiments conducted for several substrates revealed a constant product ratio. Vmax was constant for these substrates, and the overall rate of hydrolysis was increased greatly in the presence of alcohol acceptors. An enzyme-catalyzed 18O exchange between inorganic phosphate and water was detected and occurred with kcat = 4.47 x 10(-3) s-1 at pH 5.0, 37 degrees C. These results were all consistent with the existence of a phosphoenzyme intermediate in the catalytic pathway and with the breakdown of the intermediate being the rate-limiting step. The true Michaelis binding constant Ks = 6.0 mM, the apparent Km = 0.38 mM, and the rate constants for phosphorylation (k2 = 540 s-1) and dephosphorylation (k3 = 36.5 s-1) were determined under steady-state conditions with p-nitrophenyl phosphate at pH 5.0 and 37 degrees C in the presence of phosphate acceptors. The energies of activation for the enzyme-catalyzed hydrolysis at pH 5.0 and 7.0 were 13.6 and 14.1 kcal/mol, respectively. The activation energy for the enzyme-catalyzed medium 18O exchange between phosphate and water was 20.2 kcal/mol. Using the available equilibrium and rate constants, an energetic diagram was constructed for the enzyme-catalyzed reaction.     

Enzymatic studies on a cellulase system of Trichoderma viride. IV. Purification and properties of a less-random type cellulase.

A cellulase [EC 3.2.1.4] component was purified from a crude cellulase preparation of Trichoderma viride (Meicelase) by consecutive column chromatography procedures, and was designated as cellulase III. The enzyme was homogeneous on polyacrylamide gel disc electrophoresis. The molecular weight of the enzyme was estimated to be about 45,000 by gel filtration. The optimum pH and temperature of the enzyme were pH 4.5-5.0 and 50 degrees, respectively. The enzyme was stable over the range of pH 4.5-7.5 at 4 degrees for 24 hr, and retained 40% of the original carboxymethylcellulose-saccharifying activity after heating at 100 degrees for 10 min. The enzyme was completely inactivated by 1 mM Hg2+, and partially by 1 mM Ag+ and Cu2+. The enzyme was characterized as a less-random type cellulase on the basis of its action on carboxymethylcellulose. The enzyme split cellohexaose, retaining the beta-configuration of the anomeric carbon atoms in the hydrolysis products. The Km values of cellulase III for cellooligosaccharides decreased in parallel with increase of the chain length of the substrates, while Vmax values showed a tendency to increase. The enzyme produced predominantly cellobiose and glucose from various cellulosic substrates as well as from higher cellooligosaccharides. Cellulase III preferentially attacked the aglycone linkage of p-nitrophenyl beta-D-cellobioside. The enzyme was found to catalyze the rapid synthesis of cellotetraose from cellobiose (condensation action).     

Purification and characterization of alpha-L-fucosidase from Bacillus circulans grown on porcine gastric mucin.

Bacillus circulans isolated from soil was found to produce two types of alpha-L-fucosidase differing in substrate specificity. One was able to liberate L-fucose from porcine gastric mucin (PGM), but not from artificial substrates, including p-nitrophenyl and methyl alpha-L-fucosides, while the other acted not on PGM but on p-nitrophenyl alpha-L-fucoside. The production of the former enzyme was enhanced about 150 times as much by PGM added to the medium as by glucose. The alpha-L-fucosidase acting on PGM was purified from the culture fluid obtained with PGM medium by ammonium sulfate fractionation and subsequent column chromatography. The purified enzyme was found to be homogeneous by PAGE and its molecular weight was estimated to be approximately 285,000. The optimum pH was found to be 5.5 to 6.5 and the stable pH range was 4.5 to 9.0. The enzyme decomposed various blood group O(H) active substances such as PGM, human milk and human saliva, and moreover acted on A-, B-, and O-erythrocytes. The enzyme was shown to cleave alpha-(1----2)-, (1----3)-, and (1----4)-L-fucosidic linkages in various glycoproteins and oligosaccharides, but failed to hydrolyze alpha-(1----6)-L-fucosic linkages in 6-O-alpha-L-fucopyranosyl-N-acetylglucosamine and intact bovine thyroglobulin.     

Isolation and characterization of acylneuraminate cytidylyltransferase from frog liver

Frog liver (Rana esculenta) is a rich source of acylneuraminate cytidylyltransferase. The soluble enzyme was purified 250-fold almost to purity with 25% yield and a specific activity of 9 mkat/kg protein (0.54 U/mg protein) using DEAE Sephadex and Sepharose 6B chromatography, followed by preparative polyacrylamide gel electrophoresis. The molecular weight of the cytidylyltransferase was determined to be 163 000 with the aid of Sepharose 6B chromatography and gel electrophoresis, with or without dodecyl sulphate or urea. No subunits were found. The isoelectric point of the enzyme is at pH 6. Optimum reaction rate was observed at pH 9, 37 degrees C, 50mM Mg2 or Ca2 and ImM mercaptoethanol. The Km values for N-acetylneuraminic acid, N-glycoloylneuraminic acid and CTP are 1.6mM, 2.3 mM and 0.6mM, respectively. O-Acetylated sialic acids are inactive with the cytidylyltransferase from frog liver. Enzyme activity can be inhibited by SH reagents and CMP (Ki = 0.5mM).     

A neutral lipase applicable in biodiesel production from a newly isolated Streptomyces sp. CS326

In an attempt to isolate a biocatalyst able to catalyze biodiesel production from microbial source, Streptomyces sp. CS326 was screened from hundreds of soil isolates collected from various parts of Korea. In 16S rRNA sequence analysis, the strain showed high degree of similarity with Streptomyces xanthocidicus (99.79%); therefore, it is classified as Streptomyces sp. CS326. An extracellular lipase produced by the strain (LP326) was purified using a single step gel permeation chromatography on Sepharose CL-6B. Molecular weight of LP326 was estimated to be 17,000 Da by SDS-PAGE. The activity was optimum at 40 °C and pH 7.0 and was stable at pH 5.0-8.0 and below 50 °C. It preferred p-nitrophenyl palmitate (C16), a long chain substrate; and K (m) and V (max) for the substrate were determined to be 0.24 mM and 4.6 mM/min mg, respectively. First 10 N-terminal amino acid sequences were APDLVALQSE, which are different from so far reported lipases. LP326 catalyzed biodiesel production using methanol and various oils; therefore, the enzyme can be applicable in the field of biofuel.     

Purification and properties of an ethanolamine-serine base exchange enzyme of rat brain microsomes

The activity of an ethanolamine and serine base exchange enzyme of rat brain microsomes was copurified to near homogeneity. The purification sequence involved detergent solubilization, Sepharose 4B column chromatography, phenyl-Sepharose 4B column chromatography, glycerol gradient sedimentation, and agarose-polyacrylamide gel electrophoresis under non-denaturing conditions. The ratio of the ethanolamine and serine base exchange activities remained almost constant during purification, and both enzyme activities were enriched 25-fold over the initial microsomal suspension. The final enzyme preparation which contained both enzyme activities showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel, having an apparent molecular mass of about 100 kDa. Serine inhibited the ethanolamine incorporation by this preparation and ethanolamine inhibited the serine incorporation. The competitive nature of this inhibition was apparent from Lineweaver-Burk plots, suggesting that the enzyme catalyzes the incorporation of both ethanolamine and serine into their corresponding phospholipids. The Km and Ki values for ethanolamine were quite similar, being 0.02 and 0.025 mM, respectively. The Km and Ki values for serine were also quite similar being 0.11 and 0.12 mM, respectively. The pH optimum was the same at 7.0 with both substrates. The optimum Ca2+ concentration was 8 mM for serine incorporation.     

Affinity chromatographic purification of beta-glucosidase of Candida gulliermondii.

A beta-glucosidase was isolated from Candida guilliermondii, a yeast capable of growth on cellobiose. The enzyme was partially purified by treatment with polyethyleneimine and ammonium sulfate precipitation. Further purification was achieved by affinity chromatography using a Sepharose 4B matrix to which oxidized salicin was coupled through adipic dihydrazide. The final product was a 12.5-fold purification of the crude extract with a recovery of 27% of the initial enzyme activity. Polyacrylamide disc electrophoresis of the purified enzyme gave a single band. A km of 1.25 x 10(-4)M was obtained using p-nitrophenyl beta-D-glucopyranoside as the substrate. The optimum pH for enzyme activity was 6.8. Maximum activity was observed at temperature of 37 degrees C. Enzyme activity was completely inhibited by Hg++, Pb++, and Zn++ ions. The molecular weight of the enzyme is 48,000 as estimated by sucrose density gradient centrifugation.     

Properties and kinetics of a neutral beta-galactosidase from rabbit kidney

A neutral beta-galactosidase has been purified by concanavalin A-Sepharose affinity chromatography, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and hydroxylapatite chromatography. The enzyme was purified 126-fold with a yield of about 21%. This form has a neutral optimal pH (7.5) and it is located in the cytosolic fraction. It shows a wide pH stability from pH 4.5 to 8.0, but it is very unstable at low pH values. Its isoelectric point is 4.9 and this value does not change on neuraminidase treatment. The estimated molecular weight was 47 000. The neutral form shows beta-D-galactosidase, beta-D-fucosidase and beta-D-glucosidase activities, all of them associated in a single peak in all the purification steps. p-Nitrophenyl beta-D-galactosides, p-nitrophenyl beta-D-fucosides and p-nitrophenyl beta-D-glucosides competed fully for a common active site in mixed-substrate experiments. Using gamma-D-galactonolactone as competitive inhibitor the Ki values were always coincident for the three activities. The effect of NaCl, methyl mannoside and some sugars (fucose, galactose and glucose) was studied.     

Purification and properties of a secreted and developmentally regulated alpha-L-fucosidase from Dictyostelium discoideum.

During its development the eukaryotic microorganisms Dictyostelium discoideum secretes an alpha-L-fucosidase (EC 3.2.1.51). In cells of the growth phase almost no alpha-L-fucosidase activity is detectable. The activity increases steadily up to the aggregation stage and accumulates also in the extracellular medium. The developmental regulation is mediated by pulsatile cAMP signals. The alpha-L-fucosidase was purified from extracellular medium. The isolation procedure started with concentration of the enzyme by batchwise anion-exchange chromatography and ammonium sulfate precipitation, followed by Sephacryl S-300 gel filtration and further purification by fast protein liquid chromatography on Mono Q, phenyl-Superose, and finally Superose 12. The purified preparation was found to be essentially free of activities of six other glycosidases also secreted by D. discoideum. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme showed one major band with an apparent molecular mass of 62 kilodalton. Gel filtration of the enzyme on a Superose 12 column was consistent with an active monomer. A monoclonal antibody was produced, which recognizes a carbohydrate epitope shared by all lysosomal enzymes in D. discoideum. The pH optimum of the alpha-L-fucosidase is at 3.7. The apparent Michaelis constant for p-nitrophenyl alpha-L-fucoside as substrate is 1.2 mM. The enzyme catalyzes preferentially the hydrolysis of alpha 1----6GlcNAc but also of alpha 1----2Gal and alpha 1----3Glc fucosyl linkages.     

Partial purification and properties of a rat brain phospholipase.

At least 90% of a membrane-bound phospholipase D was solubilized by extraction of freeze-dried rat brain with 0.8% Miranol H2M and 0.5% cholate. The bulk of base exchange reaction enzymes remained firmly bound to the particulate fraction under these conditions. The phospholipase D specific activity was enriched 240-fold by a purification protocol employing ammonium sulfate precipitation, and both Sepharose 4B and DEAE-cellulose column chromatography. The approximate molecular weight of the partially purified enzyme was calculated to be 200,000 based upon the elution profile from Sepharose 4B and Sephadex G-200 columns. The optimum pH was 6.0, and Km values for phosphatidylcholine and phosphatidylethanolamine were 0.75 mM and 0.91 mM, respectively. The enzyme activity was not dependent on the presence of divalent cation although Ca2+ and Fe2+ showed stimulatory effects.     

Purification and properties of a novel thermostable galacto-oligosaccharide-producing beta-galactosidase from Sterigmatomyces elviae CBS8119.

A thermostable beta-galactosidase which catalyzed the production of galacto-oligosaccharide from lactose was solubilized from a cell wall preparation of Sterigmatomyces elviae CBS8119. The enzyme was purified to homogeneity by means of chromatography on DEAE-Toyopearl, Butyl-Toyopearl, Chromatofocusing, and p-aminobenzyl 1-thio-beta-D-galactopyranoside agarose columns. The molecular weight of the purified enzyme was estimated to be about 170,000 by gel filtration with a Highload-Superdex 200pg column and 86,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its isoelectric point, determined by polyacrylamide gel electrofocusing, was 4.1. The optimal temperature for enzyme activity was 85 degrees C. It was stable at temperatures up to 80 degrees C for 1 h. The optimal pH range for the enzyme was 4.5 to 5.0, it was stable at pH 2.5 to 7.0, and its activity was inhibited by Hg2+. The Km values for o-nitrophenyl-beta-D-galactopyranoside and lactose were 9.5 and 2.4 mM, respectively, and the maximum velocities for these substrates were 96 and 240 mumol/min per mg of protein, respectively. In addition, this enzyme possessed a high level of transgalactosylation activity. Galacto-oligosaccharides, including tri- and tetrasaccharides, were produced with a yield, by weight, of 39% from 200-mg/ml lactose.     

Beta-D-glycosidase activities of Humicola grisea: biochemical and kinetic characterization of a multifunctional enzyme

A beta-D-glycosidase activity was purified from mycelium of Humicola grisea var. thermoidea grown on avicel as the main carbon source. The purified enzyme was a glycoprotein and migrated as a single polypeptide band on polyacrylamide gel electrophoresis under native or denaturing conditions. The apparent molecular weight of the enzyme was estimated to be 55 kDa by gel filtration and SDS-PAGE. The enzyme was active against o-nitrophenyl beta-D-galactoside; p-nitrophenyl beta-D-glucoside, p-nitrophenyl beta-D-fucoside, lactose and cellobiose, PNP fucoside (synthetic substrate) and cellobiose (natural substrate) being the best utilized. A comparison of the properties of beta-D-galactosidase, beta-D-glucosidase and beta-D-fucosidase showed that three activities exhibited similar pH and temperature optima and the same thermostability. The hydrolysis rate of substrate mixtures suggests that the enzyme possesses a common catalytic site for all the substrates assayed.